Mutations in the glucocerebrosidase (GBA) gene have been associated with the development of Parkinson's disease (PD). An induced pluripotent stem cell (iPSC) line was generated from a 60-year old patient diagnosed with PD and carrying a new mutation variant p.R301C in GBA. Using non-integrating Sendai virus-based technology, we utilized OCT3/4, SOX2, c-MYC and KLF4 transcription factors to reprogram skin fibroblasts into iPSCs. The generated iPSC line retained the mutation, displayed expression of common pluripotency markers, differentiated into the three germ layers, and exhibited normal karyotype. The iPSC line can be further used for studying PD pathogenesis.We thank AnnaKarin Olden, Anna Hammarberg and Marianne Juhlin, for their technical support. We are also thankful to the 'Cell Line and DNA Biobank from Patients affected by Genetic Diseases' (Istituto G. Gaslini, Genova, Italy) and the 'Parkinson Institute Biobank, members of the Telethon Network of Genetic Biobanks (http://biobanknetwork.telethon.it; project no. GTB12001) funded by Telethon Italy, for providing fibroblast samples. This work was supported by the Strategic Research Environment MultiPark at Lund University, the Swedish Research Council (grant 2015-03684 to LR), Finnish Cultural Foundation (grant 00161167 to YP), Portuguese Foundation for Science and Technology for the doctoral fellowship - PDE/BDE/113598/2015 to AM and IF Starting and Development Grant to LP and AJS (IF/01079/2014 and IF/00111/2013)

Azevedo, Carla

Chumarina, Margarita

Collin, Anna

Goldwurm, Stefano

Gustavsson, Nadja

Klementieva, Oxana

Marote, Ana

Pinto, Luísa

Pomeshchik, Yuriy

Roybon, Laurent

Russ, Kaspar

Salgado, A. J.

Savchenko, Ekaterina

Stem Cell Research

Mutations in the glucocerebrosidase (GBA) gene have been associated with the development of Parkinson's disease (PD). An induced pluripotent stem cell (iPSC) line was generated from a 60-year old patient diagnosed with PD and carrying a new mutation variant p.R301C in GBA. Using non-integrating Sendai virus-based technology, we utilized OCT3/4, SOX2, c-MYC and KLF4 transcription factors to reprogram skin fibroblasts into iPSCs. The generated iPSC line retained the mutation, displayed expression of common pluripotency markers, differentiated into the three germ layers, and exhibited normal karyotype. The iPSC line can be further used for studying PD pathogenesis

Pinto, Luisa

Salgado, António J.

Lund University Publications

Generation of an induced pluripotent stem cell line (CSC-46) from a patient with Parkinson's disease carrying a novel p.R301C mutation in the GBA gene

Universidade do Minho: RepositoriUM

Contents lists available at ScienceDirectStem Cell Researchjournal homepage: www.elsevier.com/locate/scrLab Resource: Single Cell LineGeneration of an induced pluripotent stem cell line (CSC-46) from a patientwith Parkinson's disease carrying a novel p.R301C mutation in the GBA geneNadja Gustavssona, Ana Maroteb,c, Yuriy Pomeshchikd,e, Kaspar Russd,e, Carla Azevedod,e,Margarita Chumarinad,e, Stefano Goldwurmf, Anna Colling, Luisa Pintob,c, António J. Salgadob,c,Oxana Klementievaa, Laurent Roybond,e,⁎, Ekaterina Savchenkod,eaMedical Microspectroscopy, Department of Experimental Medical Science, Lund University, Lund, Swedenb Life and Health Sciences Research Institute (ICVS), School of Medicine, University of Minho, Braga, Portugalc ICVS/3B's, PT Government Associate Laboratory, Braga/Guimarães, Portugald Stem Cell Laboratory for CNS Disease Modeling, Department of Experimental Medical Science, BMC D10, Lund University, Lund, Swedene Strategic Research Area MultiPark, Lund Stem Cell Center, Lund University, Lund, Swedenf Parkinson Institute, Istituti Clinici di Perfezionamento, Milan, Italyg Department of Clinical Genetics and Pathology, Office for Medical Services, Division of Laboratory Medicine, Lund, SwedenA B S T R A C TMutations in the glucocerebrosidase (GBA) gene have been associated with the development of Parkinson's disease (PD). An induced pluripotent stem cell (iPSC) linewas generated from a 60-year old patient diagnosed with PD and carrying a new mutation variant p.R301C in GBA. Using non-integrating Sendai virus-basedtechnology, we utilized OCT3/4, SOX2, c-MYC and KLF4 transcription factors to reprogram skin fibroblasts into iPSCs. The generated iPSC line retained the mutation,displayed expression of common pluripotency markers, differentiated into the three germ layers, and exhibited normal karyotype. The iPSC line can be further usedfor studying PD pathogenesis.Resource tableUnique stem cell line i-dentifierULUNDi007-AAlternative name(s) ofstem cell lineCSC-46 LInstitution Stem Cell Laboratory for CNS Disease Modeling,Department of Experimental Medical Science, LundUniversityContact information ofdistributorLaurent Roybon, Laurent.Roybon@med.lu.seType of cell line iPSCsOrigin HumanAdditional origin info Age of patient: 60Sex of patient: MaleCell Source Skin fibroblastsClonality ClonalMethod of reprogram-mingSendai virus mediated delivery of OCT3/4, SOX2, c-MYCand KLF4Genetic Modification No modificationType of Modification N/AAssociated disease Parkinson's diseaseGene/locus GBA (MIM # 606463) on chromosome 1q22Method of modification N/AN/AName of transgene or r-esistanceInducible/constitutive s-ystemN/ADate archived/stock da-teN/ACell line repository/ba-nkN/AEthical approval Parkinson Institute Biobank (part of the Telethon GeneticBiobank Network http://biobanknetwork.telethon.it/): approved by Ethics Committee “Milano AreaC″ (http://comitatoeticoareac.ospedaleniguarda.it/) on the 26/06/2015, Numero Registro dei pareri:370–062015.Reprogramming: 202100–3211 (delivered by Swedishwork environment Arbetsmiljöverket).Resource utilityGBA mutations are associated with the development of Parkinson'sdisease (PD). The CSC-46 iPSC line was generated from a PD patientcarrying a new mutation variant p.R301C in the GBA gene. This iPSCline can be used to explore the association between PD and GBA mu-tation in disease modeling studies.https://doi.org/10.1016/j.scr.2018.101373Received 28 November 2018; Accepted 17 December 2018⁎ Corresponding author.E-mail address: Laurent.roybon@med.lu.se (L. Roybon).Stem Cell Research 34 (2019) 101373Available online 26 December 20181873-5061/ © 2019 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).TResource detailsGBA encodes the enzyme glucocerebrosidase (GBA), a lysosomalhydrolase that digests glycolipids and which is deficient in Gaucher'sdisease, a recessive lysosomal storage disorder. Mutations in GBA arelinked to an increased risk of developing PD (Sidransky and Lopez,2012) and PD patients with identified heterozygous GBA mutationspresent with early-onset motor symptoms together with cognitive im-pairment (Sidransky and Lopez, 2012; Mata et al., 2016). Several mu-tations have been identified in GBA. For example, p.N370S and p.L444Pare most frequently described in genetic screening studies (Asseltaet al., 2014). Here we report the generation of an iPSC line (clone CSC-46 L) from a PD patient carrying a new mutation variant p.R301C inGBA. This line can be used to decipher molecular mechanisms under-lying p.R301C GBA associated PD.To generate the CSC-46 iPSC line, skin fibroblasts were collected bypunch skin biopsy from a 60-year old male PD patient and repro-grammed using CytoTune™-iPS 2.0 Sendai Reprogramming Kit. Briefly,fibroblasts were seeded on a 12-well plate (75.000 cells/well) and twodays later four separate vectors carrying genes OCT-3/4, KLF-4, SOX-2and c-MYC were delivered to the cells to induce pluripotency. At day 7post-transduction, the cells were re-seeded onto mouse embryonic fi-broblasts (MEF)-feeder layer and expanded until an embryonic stem cell(ES)-like morphology was observed. At day 28, several colonies werecollected and expanded as single clones for 7 days. Three clones (CSC-46F, CSC-46 K and CSC-46 L) were selected for further expansion andkaryotype analysis. Here, we present the detailed characterization ofclone CSC-46 L (Table 1) using the methods we previously described(Holmqvist et al., 2016).The generated iPSC line CSC-46 L displayed typical colony shapeand morphology when grown on mouse fibroblast feeders (Fig. 1A). Thecolonies expressed the common nuclear and cell surface pluripotencymarkers, Oct4, Nanog, TRA1–81 and SSEA4 (Fig. 1B) as well as alkalinephosphatase (ALP) activity (Fig. 1C). In addition flow cytometry con-firmed that> 92% of the iPSCs were positive for the pluripotent cellmarker SSEA4 (Fig. 1D). The elimination of the Sendai virus was de-monstrated with immunohistochemistry analysis at passage 7 (Fig. 1E).The generated iPSC line had normal karyotype (Fig. 1F). The presenceof the p.R301C mutation in GBA gene was confirmed by DNA sequenceanalysis (Fig. 1G). Immunocytochemistry for endodermal marker alpha-fetoprotein (AFP), the mesodermal marker smooth muscle actin (SMA),and the ectodermal marker beta-III-tubulin revealed formation of allthree germ layers from the embryoid bodies (EBs) generated from theCSC-46 L line (Fig. 1H). DNA fingerprinting showed genetic equiv-alency to parental fibroblasts, thus confirming the identity of the gen-erated iPSC line. Finally, standardised qPCR test showed absence ofmycoplasma contamination in the generated iPSC line.Materials and methodsFibroblast cultureHuman dermal fibroblasts were obtained by punch skin biopsy froma PD patient carrying GBA mutation after written informed consent.Fibroblasts were cultured and expanded in culture medium containingDMEM media (ThermoFisher Scientific), 10% fetal bovine serum and1% Penicillin-Streptomycin. Cells were passaged with 0.05% trypsin(Invitrogen).iPSC generation and expansionFor reprogramming, fibroblasts were transduced using the threevector preparations (MOI=5, 5, 3) included in the CytoTune™-iPS 2.0Sendai Reprogramming Kit (Thermo Fisher Scientific). The medium wasreplaced daily for 7 days, after which the cells were re-seeded onto ir-radiated mouse embryonic fibroblasts (MEF) feeder cells. From the day8 and until day 28, the cells were cultured in WiCell medium composedof advanced DMEM/F12 (Thermo Fisher Scientific), 20% Knock-OutSerum Replacement (v/v, Thermo Fisher Scientific), 2 mM L-glutamine(Thermo Fisher Scientific), 1% non-essential amino acids (NEAA, v/v,(v/v, Thermo Fisher Scientific) and 0.1mM β-mercaptoethanol (Sigma-Aldrich), supplemented with 20 ng/mL FGF2 (Thermo FisherScientific). On day 28, several single colonies were picked and re-seeded to fresh MEFs, on a 24-well plate. Seven days later, three in-dividual clones were randomly selected and re-plated on a 6-well platefor further expansion. Cells were passaged weekly and re-plated onappropriate cell culture surface for characterization assays (Table 1).ImmunocytochemistryThe cells were fixed with 4% paraformaldehyde (PFA) for 15min atroom temperature (RT) followed by blocking and permeabilization for1 h at RT with PBS containing 10% donkey serum and 0.1% TritonX-10(Sigma). Then the cells were incubated overnight at +4 °C with theprimary antibodies (Table 2) diluted in the blocking buffer followed byincubation with secondary antibodies in the dark for 1 h at RT. DAPI(1:10000) was used for nuclei counterstaining. Image acquisition wereperformed on inverted epifluorescence microscope LRI – Olympus IX-73.Alkaline phosphatase activityAlkaline phosphatase staining was performed using AlkalinePhosphatase Staining Kit (Stemgent, MA) according to the manufac-turer's protocol.Table 1Characterization and validation.Classification Test Result DataMorphology Photography Visual record of the line: Normal morphology Fig. 1 panel APhenotype Immunocytochemistry Positive staining for pluripotency markers: Oct4, Nanog, TRA1–81 andSSEA4Fig. 1 panel BAlkaline phosphatase activity Visible activity Fig. 1 panel CFlow cytometry 92.8% SSEA4 Fig. 1 panel DGenotype Karyotype (G-banding) and resolution Normal karyotype 46,XY (300–400 bands resolution in average) Fig. 1 panel FIdentity STR analysis matched with parental fibroblasts STR analysisMutation analysis (IFAPPLICABLE)Sequencing Heterozygous p.R301C mutation in GBA Fig. 1 panel GMicrobiology and virology Mycoplasma Mycoplasma testing by RT-PCR. Negative. Not shown but availablewith authorDifferentiation potential Embryoid body formation Spontaneous EB formation and positive staining for smooth muscleactin (SMA), beta-III-tubulin (BIIITub) and α-fetoprotein (AFP)Fig. 1 panel HDonor screening (OPTIONAL) HIV 1+2 Hepatitis B, Hepatitis C N/A N/AGenotype additional info(OPTIONAL)Blood group genotyping N/A N/AHLA tissue typing N/A N/AN. Gustavsson et al. Stem Cell Research 34 (2019) 1013732In vitro differentiation by embryoid body (EB) formationiPSCs were plated on low-attachment 24-well plates and grown asembryoid bodies (EBs) for 2 weeks in WiCell medium supplementedwith 20 ng/mL FGF2. For subsequent spontaneous differentiation, EBswere seeded on a 0.1% gelatin-coated 96-well plate in DMEM mediacontaining 10% fetal bovine serum and 1% Penicillin-Streptomycin.Media was changed every 2–3 days. After 2 weeks, the cells were fixedand stained for markers against three germ layers (Table 2).Karyotype analysisThe G-banding analysis was performed at 300–400 band resolutionin a clinical diagnostic setting after 11 passages.Fig. 1. Characterization of the iPSC line CSC 46L.N. Gustavsson et al. Stem Cell Research 34 (2019) 1013733Mutation sequencingGenomic DNA was extracted from fibroblasts and iPSCs with the useof conventional lysis buffer composed of 100mM Tris (pH 8.0), 200mMNaCl, 5 mM EDTA, 1.5 mg/mL Proteinase K, and 0.2% SDS in distilledautoclaved water. Direct DNA sequencing (Macrogen Europe,Amsterdam, The Netherlands) confirmed the presence of the p.R301Cmutation in GBA. Primers used for amplification and directed sequen-cing of GBA around the mutation sites are listed in Table 2.DNA fingerprintingDNA fingerprinting analysis was performed by the IdentiCell STRprofiling service (Department of Molecular Medicine, Aarhus UniversityHospital, Skejby, Denmark).Mycoplasma detectionAbsence of mycoplasma contamination was confirmed by real-timePCR analysis at GATC Biotech AG (European Genome and DiagnosticsCentre, Konstanz, Germany).AcknowledgementsWe thank AnnaKarin Oldén, Anna Hammarberg and MarianneJuhlin, for their technical support. We are also thankful to the ‘Cell Lineand DNA Biobank from Patients affected by Genetic Diseases’ (IstitutoG. Gaslini, Genova, Italy) and the ‘Parkinson Institute Biobank, mem-bers of the Telethon Network of Genetic Biobanks (http://biobanknetwork.telethon.it; project no. GTB12001) funded byTelethon Italy, for providing fibroblast samples. This work was sup-ported by the Strategic Research Environment MultiPark at LundUniversity, the Swedish Research Council (grant 2015-03684 to LR),Finnish Cultural Foundation (grant 00161167 to YP), PortugueseFoundation for Science and Technology for the doctoral fellowship -PDE/BDE/113598/2015 to AM and IF Starting and Development Grantto LP and AJS (IF/01079/2014 and IF/00111/2013).ReferencesAsselta, R., Rimoldi, V., Siri, C., Cilia, R., Guella, I., Tesei, S., Soldà, G., Pezzoli, G., Duga,S., Goldwurm, S., 2014. Glucocerebrosidase mutations in primary parkinsonism.Parkinsonism Relat. Disord. 20 (11), 1215–1220.Holmqvist, S., Lehtonen, S., Chumarina, M., Puttonen, K., Azevedo, C., Lebedeva, O.,Ruponen, M., Oksanen, M., Djelloul, M., Collin, A., et al., 2016. Creation of a libraryof induced pluripotent stem cells from parkinsonian patients. NPJ. Parkinsons Dis. 2,16009.Mata, I.F., Leverenz, J.B., Weintraub, D., Trojanowski, J.Q., Chen-Plotkin, A., VanDeerlin, V.M., Ritz, B., Rausch, R., Factor, S.A., Wood-Siverio, C., et al., 2016. GBAvariants are associated with a distinct pattern of cognitive deficits in Parkinson dis-ease. Mov. Dis: Offl. J. Mov. Dis. Soc. 31 (1), 95–102.Sidransky, E., Lopez, G., 2012. The link between the GBA gene and parkinsonism. Lancet.Neurol. 11, 986–998.Table 2Reagents details.Antibodies used for immunocytochemistry/flow-cytometryAntibody Dilution Company Cat # and RRIDPluripotency Markers Mouse anti-Oct4 1:200 Millipore Cat# MAB4401, RRID:AB_2167852PE-conjugated mouse anti-human Nanog 1:200 BD Biosciences Cat# 560483, RRID:AB_1645522Mouse anti- TRA-1-81 1:200 Thermo Fisher Scientific Cat# 41-1100, RRID:AB_2533495PE-conjugated mouse anti-SSEA4 1:200 Thermo Fisher Scientific Cat# A14766, RRID:AB_2534281Differentiation Markers Mouse anti-AFP 1:200 Sigma-Aldrich Cat# A8452, RRID:AB_258392Mouse anti-SMA 1:200 Sigma-Aldrich Cat# A2547, RRID:AB_476701Mouse anti-beta-III- tubulin 1:200 Sigma-Aldrich Cat# T8660, RRID:AB_477590Secondary antibodies Donkey anti-mouse Alexa Fluor® 488 1:400 Molecular Probes Cat# A-21202, RRID:AB_141607Donkey anti-chicken Alexa Fluor® 488 1:400 Jackson ImmunoResearch Labs Cat# 703-545-155, RRID:AB_2340375Donkey anti-mouse Alexa Fluor® 555 1:400 Thermo Fisher Scientific Cat# A-31570, RRID:AB_2536180PrimersTarget Forward/Reverse primer (5′–3′)Targeted mutation sequencing GBA TGGTCCACTTTCTTGGCCG/AGGGGAATGGTGCTCTAGGAN. Gustavsson et al. Stem Cell Research 34 (2019) 1013734

Generation of an induced pluripotent stem cell line (CSC-46) from a patient with Parkinson's disease carrying a novel p.R301C mutation in the GBA gene

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